High-risk human papillomavirus (HPV) infections are associated with a variety of carcinomas including oropharyngeal, cervical, vulvar, vaginal, anal, and penile cancers. Over 99% of cervical cancers, which are the most common cause of cancer death in young women, are associated with high-risk HPV infections. Although there are effective vaccines against the two most common cancer causing high-risk HPV types, the vaccines only protect against new infections. For this reason, efforts into HPV research to develop effective therapies are critical to treat effected individuals, which is the ultimate goal of the work in this proposal. High-risk HPV type 16 infection results in the induction of the tumor suppressor p16INK4A as a consequence of the expression of the E7 oncoprotein. Normally, the p16INK4A protein inhibits cyclin dependent kinases (CDK) 4/6 leading to an accumulation of the hypophosphorylated form of the retinoblastoma tumor suppressor pRB which results in cellular senescence; however, HPV16 positive cells continue to proliferate since the HPV E7 protein also subverts senescence through the binding and degradation of pRB. This proposal seeks to bridge the gap in knowledge by determining cellular pathways that are dysregulated by E7 leading to oncogenic stress and induction of p16INK4A. It is known that E7 expression results in the induction of the histone 3 lysine 27 (H3K27) demethylase KDM6B, which removes repressive trimethylation marks on polycomb repressed genes including p16INK4A. Removal of a single repressive mark does not necessarily render a gene transcriptionally active, and it is unknown if E7 expression changes the core set of histone marks that need to be altered when a polycomb repressed gene is activated. The second goal of this proposal is to elucidate these changes using the p16INK4A promoter as a model gene under polycomb repression because p16INK4A serves as a biomarker for HPV- associated cancers and is an important tumor suppressor that triggers oncogene-induced senescence. Moreover, our lab has demonstrated that E7 expressing cells are epigenetically reprogrammed and become addicted to expression of p16INK4A and KDM6B and promising preliminary investigations with a small molecule inhibitor of KDM6B demonstrate decreased proliferation/survival of E7 expressing cells. Therefore, identification of other epigenetic factors that regulate p16INK4A expression may provide insight into additional potential therapeutic modalities that can be tested. As several other cancers also have increased levels of p16INK4A, these results may be applicable to the treatment of other non-HPV associated cancers.